Figure 5.

LukSF dissociation and rebinding of C5a on hC5aR-expressing cells. A) Inhibition of anti-CD88 binding on hC5aR-expressing HEK cells using increasing concentrations (x axis) of S(wt) and C5a; F(wt) is used as a negative control for inhibition of anti-CD88 binding (number of biologic repeats, n = 2). The values are normalized against the maximum binding observed with only anti-CD88. B) Disengagement of hC5aR from LukSF was observed as an increase in PE-conjugated anti-CD88 binding (right y axis, indicated with bars) on S(wt)-precoated cells using increasing but sublytic concentrations (x axis, indicated with dots) of F(wt). The values are normalized against the maximum binding observed with S(wt)-incubated cells with only anti-CD88. Minimal cell lysis (percent of lysed cells, left y axis) detected in F(wt) concentrations below 3 nM (n = 3). C) Rebinding of constant amount of NT647-labeled C5a on hC5aR upon LukSF formation, analyzed by incubating S(wt)-precoated cells with increasing concentrations (x axis) of LukF mutant F(G130D) that associates with LukS but does not lead to cell lysis (n = 2). The values are normalized against the maximum binding observed with only NT647-C5a. D) Effect of LukSF-mediated cell lysis on complement activation and C5a formation on full blood measured by using C5b-9 as a marker for complement activation in plasma (n = 3). Maximal C5a formation is observed by the incubation of full blood with live S. aureus bacteria. Ecb (B, C), F(wt) (A), or S(wt) (D) is used as a negative control in the assays. Percentages of mean fluorescence intensities is shown as relative to the maximal intensity in each individual experiment (A–C). Statistical significances are calculated using Student’s t test. Error bars indicate sd.